EP0267611A2 - Antibody against interleukin-1 - Google Patents

Antibody against interleukin-1 Download PDF

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Publication number
EP0267611A2
EP0267611A2 EP87116724A EP87116724A EP0267611A2 EP 0267611 A2 EP0267611 A2 EP 0267611A2 EP 87116724 A EP87116724 A EP 87116724A EP 87116724 A EP87116724 A EP 87116724A EP 0267611 A2 EP0267611 A2 EP 0267611A2
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Prior art keywords
antibody
human
human interleukin
process according
cells
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EP87116724A
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German (de)
French (fr)
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EP0267611A3 (en
EP0267611B1 (en
Inventor
Yasukazu 35-6 Aza-Hachishimo Ohmoto
Kenji 8-7 Aza-Shichimai Nagamura
Hisakazu 8 Aza-Nibangoshi Aoki
Yoshikatsu 5-49 Aza-Ekogawa Hirai
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Otsuka Pharmaceutical Co Ltd
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Otsuka Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/245—IL-1
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells

Definitions

  • the present invention relates to antibodies to Interleukin-1 (IL-1), and more particularly to novel antibodies to human IL-1 which make it possible to immunologically purify and determine IL-1.
  • IL-1 Interleukin-1
  • Human IL-1 is produced by macrophage monocytes and many other cells and is known to exhibit various molecular forms and biological activities.
  • Presently known as human IL-1's are two kinds which are different in isoelectric point, i.e., IL-1 ⁇ and IL-1 ⁇ .
  • the primary structures of these IL-1's have been clarified. (Nature, 315 , p641 (1985); J. Exp. Med., 164 , p237 (1986); etc.)
  • bioassays are known as techniques for determining IL-1's.
  • IL-1 is determined as the biological activity of the test sample.
  • This method nevertheless is inferior in the ease of procedure and accuracy and involves the necessity of always giving consideration to some component interfering with the measurment.
  • the method has the serious drawback of being unable to determine IL-1 ⁇ and IL-1 ⁇ as distinguished from each other since they exhibit the same activity.
  • An object of the present invention is to provide antibodies to human IL-1 ⁇ and human IL-1 ⁇ .
  • Another object of the invention is to provide antibodies to human IL-1's for use in a novel method of immunologically determining human IL-1 ⁇ and IL-1 ⁇ each individually.
  • Another object of the present invention is to provide antibodies to human IL-1's capable of neutralizing or suppressing the biological activity of IL-1 and useful in curing various diseases producing an abnormally large quantity of IL-1.
  • Another object of the invention is to provide a technique for preparing these antibodies.
  • the present invention provides a monoclonal antibody to human IL-1 which is characterized in that the antibody has specific reactivity with human IL-1 ⁇ or human IL-1 ⁇ , and a process for preparing the same.
  • the invention further provides a novel method of immunoassay by which human IL-1 ⁇ and human IL-1 ⁇ can be easily determined individually as distinguished from each other with high sensitivity and high accuracy, using the antibody of the invention.
  • the use of the antibody provides a specific method of purifying the IL-1, for example, by affinity chromatography.
  • the antibody of the invention includes a type which can neutralize the biological activity of human IL-1 and which is useful for treating various diseases producing an abnormally large quantity of IL-1, such as chronic rheumatoid arthritis, thyroiditis, hepatitis, nephritis and like chronic inflammatory diseases; arteriosclerosis, kawasaki disease and like angitis; disseminated intravascular coagulation (DIC) syndrome; blood cancer, etc.
  • the antibody can suppress the accelerated activity of the human IL-1 produced by the above diseases. Therefore the antibody of the invention provide drugs useful for preventing and curing these diseases.
  • the antibody of the present invention can be prepared by using human IL-1 ⁇ or human IL-1 ⁇ as an immunizing antigen. More specifically, the present antibody can be prepared, for example, by immunizing a mammal with the antigen, fusing the plasmacytes (immune cells) of the mammal with plasmacytoma cells of a mammal to obtain hybridoma cells, cloning the cells, selecting a clone which produces the desired antibody recognizing human IL-1 ⁇ or IL-1 ⁇ , and incubating the clone.
  • Human IL-1 ⁇ or human IL-1 ⁇ for use in the above process as the immunizing antigen is not limited specifically.
  • Useful for this purpose are, for example, a culture supernatant containing human IL-1 ⁇ and/or human IL- ⁇ derived by a known in vitro method or purified standard product thereof, human IL-1 ⁇ or human IL-1 ⁇ prepared by gene engineering techniques, and a synthetic polypeptide having a portion of the amino acid sequence of such human IL-1.
  • the mammal to be immunized with the antigen is not limited specifically. However, it is desirable to select a suitable animal in view of the amenability to the fusion of its cells with the plasmacytoma cell to be used. Generally, mice and rats are advantageously usable.
  • the mammal is immunized by a usual method, for example, by intravenously, subcutaneously or intraperitoneally giving the antigen, more specifically by administering the antigen to the animal several times every 2 to 14 days at a total dose of about 100 to 500 ⁇ g/animal.
  • a usual adjuvant is usable for the administration when required. It is desirable that the immune cells to be used be spleen cells removed about 3 days after the final administration of the immunizing antigen.
  • Plasmacytoma cells of mammals as the other host cells to be fused with the immune cells are various known cell lines including myeloma cells, such as P3 (P3/X63-­Ag8) [Nature, 256 , 495-497 (1975)], P3-UI [Current Topics in Microbiology and Immunology, 81 , 1-7 (1978)], NS-1 (Eur. J. Immunol., 6 , 511-519 (1976)], MPC-11 [Cell, 8 , 405-415 (1976)], SP2/0 [Nature, 276 , 269-270 (1978)], FO [J. Immunol. Meth., 35 , 1-21 (1980)], X63.6.5.3. [J.
  • the fusion between the immune cells and plasmacytoma cells is conducted basically by a known method, such as the method of Milstein et al (Method in Enzymology, Vol. 73, p3 (1981)). More specifically, the fusion reaction is conducted, for example, in a usual nutrient medium in the presence of a usual fusion promoting agent such as polyethylene glycol (PEG) or hemagglutinating virus of Japan (HVJ). To achieve an improved fusion efficiency, auxiliary agents such as dimethyl sulfoxide can be added to the medium when so desired.
  • PEG polyethylene glycol
  • HVJ hemagglutinating virus of Japan
  • auxiliary agents such as dimethyl sulfoxide can be added to the medium when so desired.
  • the immune cells and plasmacytoma cells are used in a usual ratio. For example, immune cells are used in about 1 to about 10 times the amount of plasmacytoma cells.
  • Examples of media useful for the fusion are RPMI-­1640 medium and MEM medium which are usually used for proliferating plasmacytoma cells, and various other media which are used for incubating cells of this type. Generally it is desirable to use such media with the serum supplement, such as fetal calf serum (FCS), removed therefrom.
  • FCS fetal calf serum
  • predetermined quantities of immune cells and plasmacytoma cells are thoroughly mixed together in the medium, and a solution of PEG having an average molecular weight of about 1000 to about 6000 is admixed, as preheated to about 37°C, with the medium usually at a concentration of about 30 to about 60 W/V%. Subsequently, a suitable medium is admixed with the culture from time to time, followed by centrifuging and removal of the supernatant every time. Repetition of this procedure gives the desired hybridoma.
  • the desired hybridoma obtained is separated by incubation in a usual selection medium such as HAT medium (containing hypoxanthine, aminopterin and thymidine).
  • HAT medium containing hypoxanthine, aminopterin and thymidine.
  • the incubation with the HAT medium is conducted for a period of time, usually several days to several weeks, which is sufficient to extinguish the cells (e.g. unfused cells) other than the desired hybridoma cells.
  • the hybridoma cells obtained are then subjected to the usual limiting dilution method to retrieve the clones producing the desired antibody, followed by the production of monoclonal antibody.
  • the antibody-producing clones can be detected by various methods which are generally used for detecting antibodies, such as the ELISA method (Engvall, E., Meth. Enzymol., 70 , 419-439 (1980), plaque method, spot method, agglutination reaction method, Ouchterlony method and radioimmunoassay (RIA) ("Hybridoma Method and Monoclonal Antibodies,” published by R & D. Planning Co., Ltd., pp. 30-53, March 5, 1982).
  • the immunizing antigen is usable for the detection.
  • the hybridoma thus obtained and producing the desired monoclonal antibody which recognizes human IL-1 ⁇ or human IL-1 ⁇ can be subcultured in a usual medium and preserved for a prolonged period of time in liquid nitrogen.
  • the desired antibody of the invention can be collected from the antibody-producing hybridoma as a culture supernatant by incubating the hybridoma in the usual manner, or as the ascites of a mammal which is amenable to the proliferation of the hybridoma, by administering the hybridoma to the animal.
  • the former method is suitable for preparing the antibody with a high purity, while the latter method is suited to the quantity production of the antibody.
  • the antibody of the invention can be easily purified by a usual method such as salting out, gel filtration, affinity chromatography or the like.
  • the monoclonal antibody thus obtained according to the invention has reactivity specific to human IL-1 ⁇ or human IL-1 ⁇ .
  • the antibodies of the invention include those of the type acting to neutralize the biological activity of human IL-1. Such antibodies are suitable for specifically determining human IL-1 having biological activity. Such antibodies, particularly the antibodies capable of recognizing the IL-1 receptor binding site of IL-1 molecule are effective for curing various diseases which abnormally produce large quantity of IL-1.
  • the antibodies of the invention further include those of the type which recognizes different sites of the human IL-1 molecule, free of steric hindrance between antibodies and capable of binding to the human IL-1 molecule. These antibodies are very useful for an immunoassay, for example, by the sandwich technique.
  • the antibodies of the invention include those of the type having high reactivity especially in a liquid or solid phase system. These antibodies are very advantageously usable for liquid phase or solid phase immunoassays.
  • the invention provides a monoclonal antibody which is specific to human IL-1 ⁇ or human IL-1 ⁇ .
  • the use of this antibody provides a method of accurately determining with high sensitivity and high specificity human IL-1 ⁇ or human IL-1 ⁇ as contained, for example, in clinical samples in a low concentration, by an immunoassay.
  • Human IL-1 ⁇ (10 ⁇ g) obtained by gene recombination techniques (Biochemistry, 58 , No.8, p.840 (1986); EPO No.187991) was intraperitoneally given to a BALB/C mouse along with complete Freund's adjuvant. The interleukin was further given every 3 to 4 weeks twice at the same dose along with an incomplete adjuvant to immunize the animal. Three to four weeks thereafter, a solution of 30 ⁇ g of human IL-1 ⁇ in physiological saline was intravenously given to the animal for final immunization.
  • cell fusion was conducted in the usual manner (see, for example, Method in Enzymology, 73 , p.3 (1981)), using spleen cells collected from the immunized animal and myeloma cells (P3U1, Current Topics in Microbiology and Immunology, 81 , 1-7 (1978)) in the ratio of 10:1 and polyethylene glycol (PEG-4000).
  • the desired hybridoma cells were selected on HAT medium.
  • the supernatant was tested by an enzyme immuno­assay using a 96-well microplate coated with human IL-1 ⁇ and peroxidase-labeled goat anti-mouse globulin (product of E. Y. Lab.) to detect cells producing the desired antibodies to human IL-1 ⁇ .
  • the cells were repeatedly cloned by the limiting dilution method to obtain 7 clones producing the desired antibodies.
  • the characteristics of the antibody obtained from each of the clones were determined by the following methods.
  • Human IL-1 ⁇ adjusted to a concentration of 20 ⁇ g/ml was placed into the wells of a 96-well microplate (100 ⁇ l/well) and allowed to stand at room temperature for 1 hour. After washing the plate with PBS, BSA was applied to the plate for blocking nonspecific adsorption, and the plate was then washed with PBS-Tween 20. A dilution of hybridoma culture supernatant was placed into the well (100 ⁇ l/well), followed by reaction at 37°C for 2 hours and then by washing.
  • Peroxidase-labeled anti-mouse immune globulin product of Cappel Labs.
  • RIA/IgG represents the reactivity of the antibody of the invention (with 125I-labeled human IL-1 ⁇ ) in the liquid phase system, while EIA/IgG stands for the reactivity of the present antibody (with human IL-1 ⁇ in the form of a solid phase) in the solid phase system.
  • the hybridoma was incubated intraperitoneally in a mouse, and the IgG1 fraction collected and purified from the ascites using IgG purifying kit (MOPS Kit, product of Bio-Lad) was checked for molecular weight (which was the sum of the molecular weights of the heavy chain and the light chain as determined by SDS-PAGE).
  • IgG purifying kit MOPS Kit, product of Bio-Lad
  • Table 1 shows the characteristics thus determined.
  • N-10 Polypeptide of human IL-1 ⁇ comprising amino acids Nos.11-153 N-16 : Polypeptide of human IL-1 ⁇ comprising amino acids Nos.17-153 N-23 : Polypeptide of human IL-1 ⁇ comprising amino acids Nos.24-153 C-82 : Polypeptide of human IL-1 ⁇ comprising amino acids Nos.1-82 C-102: Polypeptide of human IL-1 ⁇ comprising amino acids Nos.1-102 C-120: Polypeptide of human IL-1 ⁇ comprising amino acids Nos.1-120 C-140: Polypeptide of human IL-1 ⁇ comprising amino acids Nos.1-140 C-144: Polypeptide of human IL-1 ⁇ comprising amino acids Nos.1-144 C-148: Polypeptide of human IL-1 ⁇ comprising amino acids Nos.1-148
  • Antibodies ANOC 201, 206 and 207 each have a recognition site in the sequence of amino acids Nos.121 to 140 of human IL-1 ⁇ , antibodies ANOC 202 and 204 in the sequence of amino acids Nos.24 to 82, antibody ANOC 203 in the sequence of amino acids Nos.83 to 102, and antibody ANOC 205 in the sequence of amino acids Nos.145 to 148.
  • Each of the antibodies of the invention was labeled with 125I by the Iodogen method to obtain a labeled antibody.
  • Each of the present antibody was caused to be adsorbed by a polystyrene bead (6.4 mm in diameter) by the physical adsorption method (Clin. Chem. Acta., 135 , 263 (1983)) to obtain an insolubilized antibody.
  • the insolubilized antibody was reacted with 20 ng of human IL-1 ⁇ in 0.5 ml of PBS solution containing 1% BSA and 0.01% thimerosal at 37°C for 2 hours.
  • the same PBS solution (0.5 ml) as above containing about 100000 cpm of the labeled antibody obtained above was added to the bead which was washed after the reaction, followed by reaction at 37°C for 2 hours with shaking.
  • the radioactivity bound to the bead was counted to determine the reactivity between the antibodies (whether or not the sandwich of insolubilized antibody-human IL-1 ⁇ -labeled antibody was formed).
  • BALB/c3T3 fibroblasts (ATCC CCL-163) almost uniformly grown over a 6-well plate (1 ⁇ 106 cells/well) were reacted at 37°C for 4 hours with 8500 cpm/well of 125I-labeled IL-1 ⁇ (at least 250 ⁇ Ci/ ⁇ g protein in specific activity, prepared by the method of Bolton and Hunter (Biochem. J., 133 , 529 (1973)) and with an antibody of the invention (anti-IL-1 ⁇ monoclonal antibody) preincubated at 37°C in D-MEM supplemented with 10% FCS. Subsequently, 50 ⁇ l of rat serum and 250 ⁇ l of 20% PEG were reacted with the reaction mixture at 4°C for 20 minutes. The plate was then centrifuged (12000 r.p.m., 15 minutes) to obtain a sediment (bound product) and a supernatant (unbound product) separated therefrom. The bound reactivity was then measured by a gamma counter.
  • Table 3 shows the results achieved by antibodies of the invention, i.e. ANOC 203 and 205.
  • Table 3 shows that ANOC 203 of the invention inhibits 125I-labeled IL-1 ⁇ from binding to the IL-1 receptor, indicating that the antibody recognizes the IL-1 receptor binding site of IL-1 ⁇ .
  • Antibody ANOC 205 of the invention has substantially no inhibition activity. This indicates that the antibody recognizes a site different from the IL-1 receptor binding site.
  • Table 4 shows the results achieved by antibodies of the invention, i.e. ANOC 203 and 205.
  • Table 4 reveals the following.
  • IL-1 ⁇ inhibits 125I-labeled IL-1 ⁇ from binding to the IL-1 receptor
  • the present antibody ANOC 203 recognizes the site where IL-1 ⁇ binds to the IL-1 receptor, inhibiting IL-1 ⁇ from binding to the receptor and thus exhibiting recovered binding inhibitory activity.
  • the present antibody ANOC 205 does not recognize the IL-1 receptor binding site for IL-1 ⁇ , apparently failing to exhibit recovered binding inhibitory activity.
  • Clones producing the desired antibody to human IL-1 ⁇ were obtained in the same manner as in Example 1 using as an immunizing antigen human IL-1 ⁇ prepared in the same manner as human IL-1 ⁇ used above (Nature, 315 , p.641 (1985)).
  • ANOC 301 an antibody of the invention against IL-1 ⁇ , was obtained from the clones. This antibody had the following characteristics.
  • Antibody production level 75 ⁇ g/ml
  • Potency RIA X3200
  • EIA X2000 No cross reactivity on IL-2, IL-1 ⁇ , GM-CSF and TNF Binding constant (kd) : 3.6 ⁇ 10 ⁇ 8 M/L
  • Neutralizing activity Active

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Abstract

This invention provides a monoclonal antibody against human interleukin-1 characterized in that the antibody has specific reactivity with human interleukin-1α or human interleukin-1β and a process for preparing the same.

Description

  • The present invention relates to antibodies to Interleukin-1 (IL-1), and more particularly to novel antibodies to human IL-1 which make it possible to immunologically purify and determine IL-1.
  • Human IL-1 is produced by macrophage monocytes and many other cells and is known to exhibit various molecular forms and biological activities. Presently known as human IL-1's are two kinds which are different in isoelectric point, i.e., IL-1α and IL-1β. The primary structures of these IL-1's have been clarified. (Nature, 315, p641 (1985); J. Exp. Med., 164, p237 (1986); etc.)
  • Extensive research has been conducted on the application of IL-1's as drugs. Attention has been directed to the determination of IL-1's especially from clinical samples for the research on various immuno-­deficiency diseases and on abnormal immune responses and for the clinical diagnosis of these diseases and abnormalities.
  • At present, bioassays are known as techniques for determining IL-1's. With this method, IL-1 is determined as the biological activity of the test sample. This method nevertheless is inferior in the ease of procedure and accuracy and involves the necessity of always giving consideration to some component interfering with the measurment. Moreover, the method has the serious drawback of being unable to determine IL-1α and IL-1β as distinguished from each other since they exhibit the same activity.
  • An object of the present invention is to provide antibodies to human IL-1α and human IL-1β.
  • Another object of the invention is to provide antibodies to human IL-1's for use in a novel method of immunologically determining human IL-1α and IL-1β each individually.
  • Another object of the present invention is to provide antibodies to human IL-1's capable of neutralizing or suppressing the biological activity of IL-1 and useful in curing various diseases producing an abnormally large quantity of IL-1.
  • Another object of the invention is to provide a technique for preparing these antibodies.
  • These objects and other features of the invention will become apparent from the following detailed description.
  • The present invention provides a monoclonal antibody to human IL-1 which is characterized in that the antibody has specific reactivity with human IL-1α or human IL-1β, and a process for preparing the same.
  • The invention further provides a novel method of immunoassay by which human IL-1α and human IL-1β can be easily determined individually as distinguished from each other with high sensitivity and high accuracy, using the antibody of the invention.
  • Since the antibody of the invention is specific to human IL-1α or human IL-1β, the use of the antibody provides a specific method of purifying the IL-1, for example, by affinity chromatography.
  • Further, the antibody of the invention includes a type which can neutralize the biological activity of human IL-1 and which is useful for treating various diseases producing an abnormally large quantity of IL-1, such as chronic rheumatoid arthritis, thyroiditis, hepatitis, nephritis and like chronic inflammatory diseases; arteriosclerosis, kawasaki disease and like angitis; disseminated intravascular coagulation (DIC) syndrome; blood cancer, etc. The antibody can suppress the accelerated activity of the human IL-1 produced by the above diseases. Therefore the antibody of the invention provide drugs useful for preventing and curing these diseases.
  • The antibody of the present invention can be prepared by using human IL-1α or human IL-1β as an immunizing antigen. More specifically, the present antibody can be prepared, for example, by immunizing a mammal with the antigen, fusing the plasmacytes (immune cells) of the mammal with plasmacytoma cells of a mammal to obtain hybridoma cells, cloning the cells, selecting a clone which produces the desired antibody recognizing human IL-1α or IL-1β, and incubating the clone.
  • Human IL-1α or human IL-1β for use in the above process as the immunizing antigen is not limited specifically. Useful for this purpose are, for example, a culture supernatant containing human IL-1α and/or human IL-β derived by a known in vitro method or purified standard product thereof, human IL-1α or human IL-1β prepared by gene engineering techniques, and a synthetic polypeptide having a portion of the amino acid sequence of such human IL-1.
  • The mammal to be immunized with the antigen is not limited specifically. However, it is desirable to select a suitable animal in view of the amenability to the fusion of its cells with the plasmacytoma cell to be used. Generally, mice and rats are advantageously usable.
  • The mammal is immunized by a usual method, for example, by intravenously, subcutaneously or intraperitoneally giving the antigen, more specifically by administering the antigen to the animal several times every 2 to 14 days at a total dose of about 100 to 500 µg/animal. A usual adjuvant is usable for the administration when required. It is desirable that the immune cells to be used be spleen cells removed about 3 days after the final administration of the immunizing antigen.
  • Plasmacytoma cells of mammals as the other host cells to be fused with the immune cells are various known cell lines including myeloma cells, such as P3 (P3/X63-­Ag8) [Nature, 256, 495-497 (1975)], P3-UI [Current Topics in Microbiology and Immunology, 81, 1-7 (1978)], NS-1 (Eur. J. Immunol., 6, 511-519 (1976)], MPC-11 [Cell, 8, 405-415 (1976)], SP2/0 [Nature, 276, 269-270 (1978)], FO [J. Immunol. Meth., 35, 1-21 (1980)], X63.6.5.3. [J. Immunol., 123, 1548-1550 (1979)], S194 [J. Exp. Med., 148, 313-323 (1978)] etc., R210 of rat [Nature, 277, 131-133 (1979)].
  • The fusion between the immune cells and plasmacytoma cells is conducted basically by a known method, such as the method of Milstein et al (Method in Enzymology, Vol. 73, p3 (1981)). More specifically, the fusion reaction is conducted, for example, in a usual nutrient medium in the presence of a usual fusion promoting agent such as polyethylene glycol (PEG) or hemagglutinating virus of Japan (HVJ). To achieve an improved fusion efficiency, auxiliary agents such as dimethyl sulfoxide can be added to the medium when so desired. The immune cells and plasmacytoma cells are used in a usual ratio. For example, immune cells are used in about 1 to about 10 times the amount of plasmacytoma cells. Examples of media useful for the fusion are RPMI-­1640 medium and MEM medium which are usually used for proliferating plasmacytoma cells, and various other media which are used for incubating cells of this type. Generally it is desirable to use such media with the serum supplement, such as fetal calf serum (FCS), removed therefrom. To effect fusion, predetermined quantities of immune cells and plasmacytoma cells are thoroughly mixed together in the medium, and a solution of PEG having an average molecular weight of about 1000 to about 6000 is admixed, as preheated to about 37°C, with the medium usually at a concentration of about 30 to about 60 W/V%. Subsequently, a suitable medium is admixed with the culture from time to time, followed by centrifuging and removal of the supernatant every time. Repetition of this procedure gives the desired hybridoma.
  • The desired hybridoma obtained is separated by incubation in a usual selection medium such as HAT medium (containing hypoxanthine, aminopterin and thymidine). The incubation with the HAT medium is conducted for a period of time, usually several days to several weeks, which is sufficient to extinguish the cells (e.g. unfused cells) other than the desired hybridoma cells. The hybridoma cells obtained are then subjected to the usual limiting dilution method to retrieve the clones producing the desired antibody, followed by the production of monoclonal antibody.
  • The antibody-producing clones can be detected by various methods which are generally used for detecting antibodies, such as the ELISA method (Engvall, E., Meth. Enzymol., 70, 419-439 (1980), plaque method, spot method, agglutination reaction method, Ouchterlony method and radioimmunoassay (RIA) ("Hybridoma Method and Monoclonal Antibodies," published by R & D. Planning Co., Ltd., pp. 30-53, March 5, 1982). The immunizing antigen is usable for the detection.
  • The hybridoma thus obtained and producing the desired monoclonal antibody which recognizes human IL-1α or human IL-1β can be subcultured in a usual medium and preserved for a prolonged period of time in liquid nitrogen.
  • The desired antibody of the invention can be collected from the antibody-producing hybridoma as a culture supernatant by incubating the hybridoma in the usual manner, or as the ascites of a mammal which is amenable to the proliferation of the hybridoma, by administering the hybridoma to the animal. The former method is suitable for preparing the antibody with a high purity, while the latter method is suited to the quantity production of the antibody.
  • When desired, the antibody of the invention can be easily purified by a usual method such as salting out, gel filtration, affinity chromatography or the like.
  • The monoclonal antibody thus obtained according to the invention has reactivity specific to human IL-1α or human IL-1β.
  • The antibodies of the invention include those of the type acting to neutralize the biological activity of human IL-1. Such antibodies are suitable for specifically determining human IL-1 having biological activity. Such antibodies, particularly the antibodies capable of recognizing the IL-1 receptor binding site of IL-1 molecule are effective for curing various diseases which abnormally produce large quantity of IL-1.
  • The antibodies of the invention further include those of the type which recognizes different sites of the human IL-1 molecule, free of steric hindrance between antibodies and capable of binding to the human IL-1 molecule. These antibodies are very useful for an immunoassay, for example, by the sandwich technique.
  • Additionally, the antibodies of the invention include those of the type having high reactivity especially in a liquid or solid phase system. These antibodies are very advantageously usable for liquid phase or solid phase immunoassays.
  • Among the antibodies of the invention, preferred examples are as follows.
    • 1) An antibody having a recognition site in the sequence of amino acids Nos.121 to 140 of human IL-1β.
    • 2) An antibody having a recognition site in the sequence of amino acids Nos.24 to 82 of human IL-1β.
    • 3) An antibody having a recognition site in the sequence of amino acids Nos.83 to 102 of human IL-1β.
    • 4 An antibody having a recognition site in the sequence of amino acids Nos.145 to 148 of human IL-1β.
  • The invention provides a monoclonal antibody which is specific to human IL-1α or human IL-1β. The use of this antibody provides a method of accurately determining with high sensitivity and high specificity human IL-1α or human IL-1β as contained, for example, in clinical samples in a low concentration, by an immunoassay.
  • The invention will be described in greater detail with reference to the following examples, which in no way limit the invention.
    • Example 1
    • Preparation of antibodies to human IL-1β
  • Human IL-1β (10 µg) obtained by gene recombination techniques (Biochemistry, 58, No.8, p.840 (1986); EPO No.187991) was intraperitoneally given to a BALB/C mouse along with complete Freund's adjuvant. The interleukin was further given every 3 to 4 weeks twice at the same dose along with an incomplete adjuvant to immunize the animal. Three to four weeks thereafter, a solution of 30 µg of human IL-1β in physiological saline was intravenously given to the animal for final immunization. Three to four days thereafter, cell fusion was conducted in the usual manner (see, for example, Method in Enzymology, 73, p.3 (1981)), using spleen cells collected from the immunized animal and myeloma cells (P3U1, Current Topics in Microbiology and Immunology, 81, 1-7 (1978)) in the ratio of 10:1 and polyethylene glycol (PEG-4000).
  • The desired hybridoma cells were selected on HAT medium. The supernatant was tested by an enzyme immuno­assay using a 96-well microplate coated with human IL-1β and peroxidase-labeled goat anti-mouse globulin (product of E. Y. Lab.) to detect cells producing the desired antibodies to human IL-1β.
  • The cells were repeatedly cloned by the limiting dilution method to obtain 7 clones producing the desired antibodies.
  • The characteristics of the antibody obtained from each of the clones were determined by the following methods.
    • (1) Subclass of antibody
  • Determined using a mouse antibody subclass detecting kit (product of Bio-Rad).
    • (2) Antibody production level
  • Expressed in terms of the amount of IgG (µg/ml) in the culture supernatant of hybridoma when the highest cell concentration was achieved.
    • (3) Potency
  • Determined by the following procedures.
    • RIA:
  • According to the Iodogen method (B.B.R.C., 80, 849-857 (1978)), 100 µl (about 10000 cpm) of ¹²⁵I-labeled human IL-1β, 100 µl of a dilution of hybridoma culture supernatant and 100 µl of PBS containing 0.01% NaN₃, 5mM EDTA and 0.1% BSA were mixed together and reacted at 4°C. To the reaction mixture were added 50 µl of normal goat serum serving as a carrier protein and 600 µl solution of 20% PEG in PBS, and the mixture was stirred, then allowed to stand in ice bath for 20 minutes and thereafter centrifuged. The radioactivity of the sediment was measured by a gamma counter. The dilution ratio of the culture supernatant resulting in the sedimentation of 15% of ¹²⁵I-labeled IL-1β was determined as RIA potency.
    • EIA:
  • Human IL-1β adjusted to a concentration of 20 µg/ml was placed into the wells of a 96-well microplate (100 µl/well) and allowed to stand at room temperature for 1 hour. After washing the plate with PBS, BSA was applied to the plate for blocking nonspecific adsorption, and the plate was then washed with PBS-Tween 20. A dilution of hybridoma culture supernatant was placed into the well (100 µl/well), followed by reaction at 37°C for 2 hours and then by washing. Peroxidase-labeled anti-mouse immune globulin (product of Cappel Labs. Inc., X5000) was then placed into the wells in an amount of 100 µl/well, followed by reaction under the same conditions as above. After washing the plate, the enzyme activity on the plate was determined by colorimetric analysis. The dilution ratio of the culture supernatant resulting in OD₄₉₂=1.0 was determined as EIA potency.
    • RIA/IgG and EIA/IgG:
  • These are the values obtained by dividing the RIA potency and EIA potency by the antibody production level.
  • RIA/IgG represents the reactivity of the antibody of the invention (with ¹²⁵I-labeled human IL-1β) in the liquid phase system, while EIA/IgG stands for the reactivity of the present antibody (with human IL-1β in the form of a solid phase) in the solid phase system.
    • (4) Molecular weight
  • The hybridoma was incubated intraperitoneally in a mouse, and the IgG₁ fraction collected and purified from the ascites using IgG purifying kit (MOPS Kit, product of Bio-Lad) was checked for molecular weight (which was the sum of the molecular weights of the heavy chain and the light chain as determined by SDS-PAGE).
    • (5) Cross reactivity
  • Determined by Western blotting (Proc. Natl. Acad. Sci., U.S.A., 76, 4350 (1979); Anal. Biochem., 112, 195 (1981)) using human recombinant proteins prepared by genetic engineering techniques. The symbol "-" represents absence of cross reactivity.
    • (6) Binding constant (kd)
  • Determined by Scatchard blot analysis.
    • (7) Neutralizing activity
  • Determined from LAF activity (J. Immunol., 116, 1466 (1976)). The mark "+" represents presence of neutralizing activity.
  • Table 1 shows the characteristics thus determined.
    Figure imgb0001
    Figure imgb0002
    • (8) Determination of binding site
  • The following fragments of human IL-1β were prepared by the method described in EPO No.187991 for preparing human IL-1β.
    • Fragments
  • N-10 : Polypeptide of human IL-1β comprising amino acids Nos.11-153
    N-16 : Polypeptide of human IL-1β comprising amino acids Nos.17-153
    N-23 : Polypeptide of human IL-1β comprising amino acids Nos.24-153
    C-82 : Polypeptide of human IL-1β comprising amino acids Nos.1-82
    C-102: Polypeptide of human IL-1β comprising amino acids Nos.1-102
    C-120: Polypeptide of human IL-1β comprising amino acids Nos.1-120
    C-140: Polypeptide of human IL-1β comprising amino acids Nos.1-140
    C-144: Polypeptide of human IL-1β comprising amino acids Nos.1-144
    C-148: Polypeptide of human IL-1β comprising amino acids Nos.1-148
  • To E. coli cells expressing human IL-1β or each of the fragments was added 100 µl of 6.25 mM Tris buffer (pH 6.8, containing 2% SDS, 5% 2ME, 10% glycerol and 0.001% BPB) to obtain a solution, which was then boiled at 100°C for 2 minutes to obtain a sample for electro­phoresis. The samples thus prepared were tested for reactivity with antibodies of the invention by Western blotting.
  • Table 2 below shows the result.
    Figure imgb0003
  • Table 2 reveals the following. Antibodies ANOC 201, 206 and 207 each have a recognition site in the sequence of amino acids Nos.121 to 140 of human IL-1β, antibodies ANOC 202 and 204 in the sequence of amino acids Nos.24 to 82, antibody ANOC 203 in the sequence of amino acids Nos.83 to 102, and antibody ANOC 205 in the sequence of amino acids Nos.145 to 148.
    • (9) Steric hindrance test of present antibodies
  • Each of the antibodies of the invention was labeled with ¹²⁵I by the Iodogen method to obtain a labeled antibody.
  • Each of the present antibody was caused to be adsorbed by a polystyrene bead (6.4 mm in diameter) by the physical adsorption method (Clin. Chem. Acta., 135, 263 (1983)) to obtain an insolubilized antibody.
  • The insolubilized antibody was reacted with 20 ng of human IL-1β in 0.5 ml of PBS solution containing 1% BSA and 0.01% thimerosal at 37°C for 2 hours. The same PBS solution (0.5 ml) as above containing about 100000 cpm of the labeled antibody obtained above was added to the bead which was washed after the reaction, followed by reaction at 37°C for 2 hours with shaking.
  • After washing the bead, the radioactivity bound to the bead was counted to determine the reactivity between the antibodies (whether or not the sandwich of insolubilized antibody-human IL-1β-labeled antibody was formed).
  • Consequently, no sandwich was formed between antibodies ANOC 201, 206 and 207, indicating that these antibodies recognize the substantially the same site of IL-1β.
  • No hindrance was found in the reaction between all the other antibodies tested (except in the reaction the antibody with itself).
    • (10) Inhibition of binding of IL-1β to IL-1 receptor on fibroblast (Test A)
  • BALB/c3T3 fibroblasts (ATCC CCL-163) almost uniformly grown over a 6-well plate (1 × 10⁶ cells/well) were reacted at 37°C for 4 hours with 8500 cpm/well of ¹²⁵I-labeled IL-1β (at least 250 µCi/µg protein in specific activity, prepared by the method of Bolton and Hunter (Biochem. J., 133, 529 (1973)) and with an antibody of the invention (anti-IL-1β monoclonal antibody) preincubated at 37°C in D-MEM supplemented with 10% FCS. Subsequently, 50 µl of rat serum and 250 µl of 20% PEG were reacted with the reaction mixture at 4°C for 20 minutes. The plate was then centrifuged (12000 r.p.m., 15 minutes) to obtain a sediment (bound product) and a supernatant (unbound product) separated therefrom. The bound reactivity was then measured by a gamma counter.
  • The activity (%) of the present antibody to inhibit IL-1β from binding to the IL-receptor on the fibroblast was calculated from the following equation.
        Inhibition activity (%) =
    Figure imgb0004
    × 100
    wherein A: radioactivity of control without the antibody of invention
    B: radioactivity nonspecifically adsorbed by the plate
    C: Radioactivity due to the use of the present antibody
  • In this test, the value A was 3895 cpm/well, and the value C was 301 cpm.
  • Table 3 shows the results achieved by antibodies of the invention, i.e. ANOC 203 and 205.
    Figure imgb0005
  • Table 3 shows that ANOC 203 of the invention inhibits ¹²⁵I-labeled IL-1β from binding to the IL-1 receptor, indicating that the antibody recognizes the IL-1 receptor binding site of IL-1β. Antibody ANOC 205 of the invention has substantially no inhibition activity. This indicates that the antibody recognizes a site different from the IL-1 receptor binding site.
    • (11) Inhibition of binding to IL-1 receptor on fibroblast (Test B)
  • Into each of wells having fibroblasts grown therein were placed ¹²⁵I-labeled IL-1α, 10 ng/ml of IL-1β and a specified quantity of an antibody of the invention, and the mixture was reacted in the same manner as in the procedure (10) above. After similarly centrifuging the plate, the radioactivity of the well was measured, and the activity (%) of the present antibody to inhibit IL-1β from binding to the IL-1 receptor on the fibroblast was calculated.
  • In this test, the value A was 8278 cpm/well, and the value C was 410 cpm.
  • Table 4 shows the results achieved by antibodies of the invention, i.e. ANOC 203 and 205.
    Figure imgb0006
  • Table 4 reveals the following. IL-1β inhibits ¹²⁵I-labeled IL-1α from binding to the IL-1 receptor, whereas the present antibody ANOC 203 recognizes the site where IL-1β binds to the IL-1 receptor, inhibiting IL-1β from binding to the receptor and thus exhibiting recovered binding inhibitory activity. The present antibody ANOC 205 does not recognize the IL-1 receptor binding site for IL-1β, apparently failing to exhibit recovered binding inhibitory activity.
    • Example 2 Preparation of antibody to human IL-1α
  • Clones producing the desired antibody to human IL-1α were obtained in the same manner as in Example 1 using as an immunizing antigen human IL-1α prepared in the same manner as human IL-1β used above (Nature, 315, p.641 (1985)). ANOC 301, an antibody of the invention against IL-1α, was obtained from the clones. This antibody had the following characteristics. Subclass      : IgG2a
    Antibody production level      : 75 µg/ml
    Potency      : RIA X3200 EIA X2000
    Cross reactivity      : No cross reactivity on IL-2, IL-1β, GM-CSF and TNF
    Binding constant (kd)      : 3.6 × 10⁻⁸ M/L
    Neutralizing activity      : Active
  • Each of four hybrid cell lines obtained in the examples has been deposited in Fermentation Research Institute, Agency of Industrial Science and Technology since November 5, 1987 under the following deposition names and numbers, respectively.
    Figure imgb0007

Claims (14)

1. A process for preparing a monoclonal antibody having specific reactivity with human interleukin-1α or human interleukin-1β, characterized by immunizing a mammal with human interleukin-1α or human interleukin-1β serving as an immunizing antigen, fusing the immune cells with plasmacytoma cells of a mammal to obtain hybridoma cells, cloning the cells, selecting a clone which produces the desired antibody recognizing human interleukin-1α or human interleukin-1β, and incubating the clone.
2. The process according to claim 1, characterized in that the monoclonal antibody possesses an activity to neutralize the biological activity of human interleukin-1.
3. The process according to claim 1, characterized in that the antibody has a recognition site in the sequence of amino acid Nos. 121 to 140 of humun interleukin-1β.
4. The process according to claim 1, characterized in that the antibody has a recognition site in the sequence of amino acid Nos. 24 to 82 of human interleukin-1β.
5. The process according to claim 1, characterized in that the antibody has a recognition site in the sequence of amino acid Nos. 83 to 102 of human interleukin-1β.
6. The process according to claim 1, characterized in that the antibody has a recognition site in the sequence of amino acid Nos. 145 to 148 of human interleukin-1β.
7. The process according to claim 1, characterized in that the antibody is produced by the hybrid cell line FERM BP-1551.
8. The process according to claim 1, characterized in that the antibody is produced by the hybrid cell line FERM BP-1552.
9. The process according to claim 1, characterized in that the antibody is produced by the hybrid cell line FERM BP-1553.
10. The process according to claim 1, characterized in that the antibody is produced by the hybrid cell line FERM BP-1554.
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DK590387D0 (en) 1987-11-11
ES2054644T3 (en) 1994-08-16
DE3785994D1 (en) 1993-07-01
KR880006357A (en) 1988-07-22
DE3785994T2 (en) 1993-11-11
EP0267611A3 (en) 1988-09-21
KR900004420B1 (en) 1990-06-25
EP0267611B1 (en) 1993-05-26
DK590387A (en) 1988-05-14

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